asking suggestions for western blot

Hi, I have been doing western blot for two month now, but still could not get satisfied result. Met couple of problems, hope can get some advice from your guys.

First, I could not get clear band for my protein standard (benchmard protein standard from invitrogen). Rather than getting several clear seperating bands, I got several big spots and some of them are merged. I only used 1ul. And I am also wondering, is Anti-His C the only one antibody I can use to detect this ladder?
Second problem is with my negative control. I cloned my gene into pYES2.1 TOPO vector, so I was using pYES2.1/V5-His/lacZ as negative control. Since my gene can not be detected by anti-His C antibody, I can only use anti-V5. Usually, I got single band for negative control when anti-His C was used. But there were multiple bands in my nagative control when I was using anti-V5. did anyone use anti-V5 antibody as well? can someone tell me the difference about these two antibodies?
Thanks in advance,

Benchmark is designed for detection by coomassie or silver staining of gels (not on membranes), and you really need to read the product manual to find out how much to load per gel (usually you will need to load at least 5 ul). You should be able to detect the ladder by Ponceau S staining the membranes.

I would be surprised if the ladder is detectable with any antibodies, unless you are using the Magicmark range of ladders instead - which are detectable with any secondary antibody as they are conjugated IgG chains.

Hi, I have been doing western blot for two month now, but still could not get satisfied result. Met couple of problems, hope can get some advice from your guys.

First, I could not get clear band for my protein standard (benchmard protein standard from invitrogen). Rather than getting several clear seperating bands, I got several big spots and some of them are merged. I only used 1ul. And I am also wondering, is Anti-His C the only one antibody I can use to detect this ladder? Second problem is with my negative control. I cloned my gene into pYES2.1 TOPO vector, so I was using pYES2.1/V5-His/lacZ as negative control. Since my gene can not be detected by anti-His C antibody, I can only use anti-V5. Usually, I got single band for negative control when anti-His C was used. But there were multiple bands in my nagative control when I was using anti-V5. did anyone use anti-V5 antibody as well? can someone tell me the difference about these two antibodies?Thanks in advance,

Penny

Hi Penny,

Why don't you use pre-stained markers? It just makes life so much easier. Have a look at Fermentas' page rulers #SM1811, #SM0671. For WB on nitrocellulose I use 1 ul, so they are very economical too.

With regards tot he antibodies, your information is a bit sparse. According to you the band in the negative using the His Ab should then be the lacZ gene? Otherwise there should be nothing. Why can't your gene be detected by the His Ab, did you remove the His-tag when you cloned it?

I use anti V5 antibody with pcDNA 3.2v5, I usually get clear defined bands after 1-5 minutes of exposure using a 1:5000 dilution on transient transfections loading ~15ug of protein. I've never seen any bands in negative controls even after a 24 hour exposure. What are your dilution rates/protein loaded/exposure times? And are these transients?